PCR has revolutionized the diagnosis of infectious diseases. Detecting and identifying microorganisms by their DNA or RNA is potentially more sensitive, more specific and accurate, and allows nearly immediate detection compared to phenotypic tests for most organisms. Fastidious organisms, perhaps many more than we are aware of, are essentially unculturable, or require enriched culture medium and excessive incubation times. Despite this potential, PCR-based testing of fungi in the clinical lab is lagging behind virus and bacteria. Lack of widely used molecular tools for identifying fungi is a shame, given the high mortality rates associated with systemic fungal infections that are often diagnosed too late. Detection of fungal infections is a challenge for PCR-based methods, due to the low numbers of cells at sampling sites, and the hard cell wall that complicates efficient cell lysis. Reviews of the literature underscore that more optimization is needed before PCR will be reliable for diagnosis of Aspergillus fumigatus and other fungi. Our long term objective is to develop rapid, quantitative and sensitive assays for detection and identification of fungal species, and ultimately their antifungal susceptibilities, directly from clinical samples. Toward this goal, our specific aims are: 1. To optimize sample processing to provide single cell detection from any clinical source. 2. To modify current methods of fungal cell lysis to achieve DNA recovery from single fungal cells in the presence of a vast excess of human cells, in a manner conducive to high-throughput processing, with minimal technician skill. 3. To explore different methods of generating species-specific primers or hybridization probes, and to compare our primers with those developed in other laboratories. 4. To extend current real time PCR capabilities for Candida albicans and Aspergillus fumigatus to other fungal species. 5. To validate the new assays on clinical samples from patients with definite or probable fungal infection. We expect that the protocols and strategies developed here for fungi will extend to other, non-culturable organisms.